We have defined a human mammary differentiation antigen using murine monoclonal antibody (MAb DF3) prepared against a membrane enriched fraction of a human breast carcinoma. This antigen has a molecular weight of 290,000, and is detectable on the cell surface of human breast carcinoma cells using a live cell radioimmunoassay and fluorescence flow cytometry. More importantly, immunoperoxidase staining with MAb DF3 distinguishes malignant and benign breast lesions via cellular distribution of reactive antigen. A cytoplasmic staining pattern has been observed with a majority of breast carcinomas, and only one of 13 fibroadenoma or fibrocystic disease specimens. In contrast, reactivity of benign breast lesions with MAb DF3 primarily occurs along apical borders. These results demonstrate that the DF3 antigen is present on apical borders of more differentiated secretory mammary epithelial cells and in the cytosol of less differentiated cells. This novel monoclonal antibody may be employed to distinguish less differentiated from better differentiated mammary carcinomas.